I was wondering whether the DNase1 treated sample can be used directly for making cDNA. I did this with Biorad iscript cDNA kit and it did not work. Looks like the buffers are not compatible. Has anyone experienced using promega Dnase1 treatment and ABI high capacity cDNA prep without further RNA purification. Any suggestion would be greatly appreciated. Thanks
Have you denatured DNase I before proceding to cDNA synthesis? I often do chloroform extraction after DNase I treatment. I never had problem
Yes did heat denaturation. I am using human primary samples. So sample is limited. I am afraid during 2nd extraction I may loose sample. Definitely that would the best. Thank you for your response.
For expression studies our lab uses the Promega RQ1 DNAse according to the protocol with no issues. However, we run a confirmation PCR to ensure our target specific DNA is degraded before moving onto cDNA synthesis. We also perform a lithium chloride RNA precipitation after the confirmation PCR. This might be a little over board, but from our bioanalyzer results the RNA quality and integrity is very good for qPCR.
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